Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16
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- Fukui, T., Ohsawa, K., Mifune, J. et al. Appl Microbiol Biotechnol (2011) 89: 1527. doi:10.1007/s00253-011-3100-2
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Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by Plac, Ptac, or PBAD derived from Escherichia coli, or promoter regions of phaC1 (PphaC) or phaP1 (PphaP) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. Plac, Ptac, PphaC, and PphaP mediated constitutive gene expression, among which Ptac was the strongest promoter. lacI-Ptac was not thoroughly functional even after addition of isopropyl-β-d-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-PBAD could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-PphaP exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-PphaP for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.